In our studies, we have continued to identify peptides derived from sequences of several known tumor-associated antigens (TAA) that are capable of eliciting either cytotoxic or helper T cell responses against tumors. Three novel T-helper epitopes that overlap or lie proximal with CTL epitopes derived from gp100 melanoma tumor antigen have been identified. Because the newly identified helper T lymphocyte (HTL) epitopes lie proximal to previously described CTL epitopes for this tumor-associated antigen, the use of peptide vaccines containing both CTL and HTL epitopes could offer therapeutic advantages over current approaches that focus solely on CTL epitopes. The new defined THL/CTL overlapping epitopes are now being tested in clinical trial. We have also explored the intracellular delivery of CTL epitope so that multi-epitope peptide vaccines can be efficiently processed and presented. We have designed Trojan antigens which are composed of CTL epitopes linked with membrane translocating peptides and found that these antigens are able to generate CTL epitopes through a TAP independent mechanism since they have the capacity to translocate into the ER and Golgi compartments. This is a simple approach to deliver peptide constructs into APC for the generation of CTL epitopes in TAP independent fashion, which could be valuable for the development of T cell vaccines by either direct invivoimmunization or invitrosensitization of dendritic cells. Recently, in collaboration with Dr. DeLeo, we have used autologous dendritic cells(DC) pulsed with p53(264-272) peptide to generate p53 peptide-specific T cells from peripheral blood of patients with squamous cell carcinoma of the head and neck (SCCHN), as well as healthy donors. The following progress was made during the past year towards development of broadly applicable p53-based cancer vaccines. Analysis that PBMC from only approximately 1 in 3 HLA-A2.1+ healthy donors or oral cancer patients are responsive exvivoto the HLA-A2.1-restricted CTL-defined wt p53264-272 epitope. We identified alternative peptide ligands (APL) of this epitope, which have enhanced immunogenicity. As a result of using these APLs, we were able to increase the frequency of generating anti-tumor CTL recognizing the parental epitope to ~ 2 of 3 donors. In addition, CD4+ T cells from PBMC of a healthy HLA-DR4+ donor were stimulated exvivowith autologous dendritic cells in the presence of recombinant human p53. This bulk population of lymphocytes showed specificity for one of eight putative HLA-DR4-binding wt p53 peptides tested, namely the p53 110-124 peptide. These effectors also were reactive in an ELISPOT assay against an oral cancer cell line that expresses HLA-DR4 molecules following treatment with IFNg, confirming that this peptide is a naturally presented, Th-defined wt p53 epitope. This finding expands the repertoire of wt p53 epitopes available for development of p53 vaccines and will permit the targeting of CTL as well as Th-defined wt p563 epitopes in the future. Presently, we are continuing work on the optimization of peptide epitopes for targeted immunotherapy, emphasizing the development of superagonist ligands in different systems. We also are continuing our studies on crystal structures of T cell receptor (TcR)/MHC class I peptide complexes having partial agonist, antagonist, and superagonist activity. In addition, we have determined that agonist altered peptide ligands (APL) have higher affinity for the AH3 TcR than antagonist and null ligands. In order to look more closely at the molecular basis of T cell xenoreactivity, in collaboration with Dr. Collins we have also solved a co-crystal structure of the AH3 T cell receptor bound to its xenoreactive ligand (HLA-A2.1 complexed with a peptide termed p1049) to 2.5 Angstroms.